Increase In Double-strand Breaks Research Articles

Histone variant H2A.Z is needed for efficient transcription-coupled NER and genome integrity in UV challenged yeast cells.

The genome of living cells is constantly challenged by DNA lesions that interfere with cellular processes such as transcription and replication. A manifold of mechanisms act in concert to ensure adequate DNA repair, gene expression, and genome stability. Bulky DNA lesions, such as those induced by UV light or the DNA-damaging agent 4-nitroquinoline oxide, act as transcriptional and replicational roadblocks and thus represent a major threat to cell metabolism. When located on the transcribed strand of active genes, these lesions are handled by transcription-coupled nucleotide excision repair (TC-NER), a yet incompletely understood NER sub-pathway. Here, using a genetic screen in the yeast Saccharomyces cerevisiae, we identified histone variant H2A.Z as an important component to safeguard transcription and DNA integrity following UV irradiation. In the absence of H2A.Z, repair by TC-NER is severely impaired and RNA polymerase II clearance reduced, leading to an increase in double-strand breaks. Thus, H2A.Z is needed for proficient TC-NER and plays a major role in the maintenance of genome stability upon UV irradiation.

Hyperthermia as a Potential Cornerstone of Effective Multimodality Treatment with Radiotherapy, Cisplatin and PARP Inhibitor in IDH1-Mutated Cancer Cells.

Mutations in the isocitrate dehydrogenase 1 (IDH1MUT) gene occur in various types of malignancies, including ~60% of chondrosarcomas, ~30% of intrahepatic cholangiocarcinomas and >80% of low-grade gliomas. IDH1MUT are causal in the development and progression of these types of cancer due to neomorphic production of the oncometabolite D-2-hydroxyglutarate (D-2HG). Intracellular accumulation of D-2HG has been implicated in suppressing homologous recombination and renders IDH1MUT cancer cells sensitive to DNA-repair-inhibiting agents, such as poly-(adenosine 5'-diphosphate-ribose) polymerase inhibitors (PARPi). Hyperthermia increases the efficacy of DNA-damaging therapies such as radiotherapy and platinum-based chemotherapy, mainly by inhibition of DNA repair. In the current study, we investigated the additional effects of hyperthermia (42 °C for 1 h) in the treatment of IDH1MUT HCT116 colon cancer cells and hyperthermia1080 chondrosarcoma cancer cells in combination with radiation, cisplatin and/or a PARPi on clonogenic cell survival, cell cycle distribution and the induction and repair of DNA double-strand breaks. We found that hyperthermia in combination with radiation or cisplatin induces an increase in double-strand breaks and cell death, up to 10-fold in IDH1MUT cancer cells compared to IDH1 wild-type cells. This vulnerability was abolished by the IDH1MUT inhibitor AGI-5198 and was further increased by the PARPi. In conclusion, our study shows that IDH1MUT cancer cells are sensitized to hyperthermia in combination with irradiation or cisplatin and a PARPi. Therefore, hyperthermia may be an efficacious sensitizer to cytotoxic therapies in tumors where the clinical application of hyperthermia is feasible, such as IDH1MUT chondrosarcoma of the extremities.

Turning coldspots into hotspots: targeted recruitment of axis protein Hop1 stimulates meiotic recombination in Saccharomyces cerevisiae.

The DNA double-strand breaks that initiate meiotic recombination are formed in the context of the meiotic chromosome axis, which in Saccharomyces cerevisiae contains a meiosis-specific cohesin isoform and the meiosis-specific proteins Hop1 and Red1. Hop1 and Red1 are important for double-strand break formation; double-strand break levels are reduced in their absence and their levels, which vary along the lengths of chromosomes, are positively correlated with double-strand break levels. How axis protein levels influence double-strand break formation and recombination remains unclear. To address this question, we developed a novel approach that uses a bacterial ParB-parS partition system to recruit axis proteins at high levels to inserts at recombination coldspots where Hop1 and Red1 levels are normally low. Recruiting Hop1 markedly increased double-strand breaks and homologous recombination at target loci, to levels equivalent to those observed at endogenous recombination hotspots. This local increase in double-strand breaks did not require Red1 or the meiosis-specific cohesin component Rec8, indicating that, of the axis proteins, Hop1 is sufficient to promote double-strand break formation. However, while most crossovers at endogenous recombination hotspots are formed by the meiosis-specific MutLγ resolvase, crossovers that formed at an insert locus were only modestly reduced in the absence of MutLγ, regardless of whether or not Hop1 was recruited to that locus. Thus, while local Hop1 levels determine local double-strand break levels, the recombination pathways that repair these breaks can be determined by other factors, raising the intriguing possibility that different recombination pathways operate in different parts of the genome.

Mechanistic insight into the repair of C8-linked pyrrolobenzodiazepine monomer-mediated DNA damage.

Pyrrolobenzodiazepines (PBDs) are naturally occurring DNA binding compounds that possess anti-tumor and anti-bacterial activity. Chemical modifications of PBDs can result in improved DNA binding, sequence specificity and enhanced efficacy. More recently, synthetic PBD monomers have shown promise as payloads for antibody drug conjugates and anti-bacterial agents. The precise mechanism of action of these PBD monomers and their role in causing DNA damage remains to be elucidated. Here we characterized the damage-inducing potential of two C8-linked PBD bi-aryl monomers in Caulobacter crescentus and investigated the strategies employed by cells to repair the same. We show that these compounds cause DNA damage and efficiently kill bacteria, in a manner comparable to the extensively used DNA cross-linking agent mitomycin-C (MMC). However, in stark contrast to MMC which employs a mutagenic lesion tolerance pathway, we implicate essential functions for error-free mechanisms in repairing PBD monomer-mediated damage. We find that survival is severely compromised in cells lacking nucleotide excision repair and to a lesser extent, in cells with impaired recombination-based repair. Loss of nucleotide excision repair leads to significant increase in double-strand breaks, underscoring the critical role of this pathway in mediating repair of PBD-induced DNA lesions. Together, our study provides comprehensive insights into how mono-alkylating DNA-targeting therapeutic compounds like PBD monomers challenge cell growth, and identifies the specific mechanisms employed by the cell to counter the same.

Abstract 2039: The role of HORMAD1 in DNA damage repair in squamous cell carcinomas

Abstract Genomic instability is a prominent hallmark of cancer, however the dynamic mechanisms that drive and sustain genomic instability remain elusive. Research demonstrates that numerous cancers with increased levels of genomic instability express meiosis genes and undergo a process called meiomitosis. Cancer meiomitosis is the orchestrated activation of both mitotic and meiotic machineries in neoplastic cells that confer a selective advantage. Specifically, meiCT (meiosis-specific cancer/testis) genes that specialize in the reductional division of germ cells in meiosis I are ectopically expressed in several cancers, particularly in squamous cell carcinomas. The meiCT gene, HORMAD1 is a meiosis specific protein that functions to ensure that a sufficient number of DSBs are formed for appropriate meiotic progression and to maintain genome integrity throughout homologous recombination. Studies show that HORMAD1 is significantly upregulated in several cancers and is regarded as a potentially important oncogene that plays a role in sustaining increased genomic instability. With the use of shRNA mediated knockdown of HORMAD1, we evaluated the effects ofHORMAD1 on genomic instability and survival in squamous cell carcinomas (SCCs). Our results demonstrate that HORMAD1 knockdown in SCC cancer cell lines leads to an increase in genomic instability denoted by an increase in double strand breaks, micronuclei formation and ploidy. Furthermore, shHORMAD1 results in increased sensitivity to etoposide treatment leading to increased centrosome formation and reduced cell survival. We also demonstrate thatHORMAD1 modulates a functional and balanced level of genomic instability by eliciting an DNA damage response through homologous recombination mechanisms that allows SCCs to thrive following DNA damage. Citation Format: Jennifer Gantchev, Amelia Martinez Villarreal, Brandon Ramchatesingh, Ivan V. Litvinov. The role of HORMAD1 in DNA damage repair in squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2039.

HTERT gene knockdown enhances response to radio- and chemotherapy in head and neck cancer cell lines through a DNA damage pathway modification

The aim of the study was to analyze the effect of hTERT gene knockdown in HNSCC cells by using novel in vitro models of head and neck cancer (HNSCC), as well as improving its personalized therapy. To obtain the most efficient knockdown siRNA, shRNA-bearing lentiviral vectors were used. The efficiency of hTERT silencing was verified with qPCR, Western blot, and immunofluorescence staining. Subsequently, the type of cell death and DNA repair mechanism induction after hTERT knockdown was assessed with the same methods, followed by flow cytometry. The effect of a combined treatment with hTERT gene knockdown on Double-Strand Breaks levels was also evaluated by flow cytometry. Results showed that the designed siRNAs and shRNAs were effective in hTERT knockdown in HNSCC cells. Depending on a cell line, hTERT knockdown led to a cell cycle arrest either in phase G1 or phase S/G2. Induction of apoptosis after hTERT downregulation with siRNA was observed. Additionally, hTERT targeting with lentiviruses, followed by cytostatics administration, led to induction of apoptosis. Interestingly, an increase in Double-Strand Breaks accompanied by activation of the main DNA repair mechanism, NER, was also observed. Altogether, we conclude that hTERT knockdown significantly contributes to the efficacy of HNSCC treatment.

Resolvase OsGEN1 Mediates DNA Repair by Homologous Recombination.

Yen1/GEN1 are canonical Holliday junction resolvases that belong to the RAD2/XPG family. In eukaryotes, such as budding yeast, mice, worms, and humans, Yen1/GEN1 work together with Mus81-Mms4/MUS81-EME1 and Slx1-Slx4/SLX1-SLX4 in DNA repair by homologous recombination to maintain genome stability. In plants, the biological function of Yen1/GEN1 remains largely unclear. In this study, we characterized the loss of function mutants of OsGEN1 and OsSEND1, a pair of paralogs of Yen1/GEN1 in rice (Oryza sativa). We first investigated the role of OsGEN1 during meiosis and found a reduction in chiasma frequency by ∼6% in osgen1 mutants, compared to the wild type, suggesting a possible involvement of OsGEN1 in the formation of crossovers. Postmeiosis, OsGEN1 foci were detected in wild-type microspore nuclei, but not in the osgen1 mutant concomitant with an increase in double-strand breaks. Persistent double-strand breaks led to programmed cell death of the male gametes and complete male sterility. In contrast, depletion of OsSEND1 had no effects on plant development and did not enhance osgen1 defects. Our results indicate that OsGEN1 is essential for homologous recombinational DNA repair at two stages of microsporogenesis in rice.

Insulin-Like Growth Factor-1–Mediated DNA Repair in Irradiated Salivary Glands Is Sirtuin-1 Dependent

Ionizing radiation is one of the most common cancer treatments; however, the treatment leads to a wide range of debilitating side effects. In patients with head and neck cancer (HNC), the surrounding normal salivary gland is extremely sensitive to therapeutic radiation, and damage to this tissue results in various oral complications and decreased quality of life (QOL). In the current study, mice treated with targeted head and neck radiation showed a significant increase in double-stranded breaks (DSB) in the DNA of parotid salivary gland cells immediately after treatment, and this remained elevated 3 h posttreatment. In contrast, mice pretreated with insulin-like growth factor-1 (IGF-1) showed resolution of the same amount of initial DNA damage by 3 h posttreatment. At acute time points (30 min to 2 h), irradiated parotid glands had significantly decreased levels of the histone deactylase Sirtuin-1 (SirT-1) which has been previously shown to function in DNA repair. Pretreatment with IGF-1 increased SirT-1 protein levels and increased deacetylation of SirT-1 targets involved in DNA repair. Pharmacological inhibition of SirT-1 activity decreased the IGF-1–mediated resolution of DSB. These data suggest that IGF-1 promotes DNA repair in irradiated parotid glands through the maintenance and activation of SirT-1.

TU‐H‐CAMPUS‐TeP3‐01: Gold Nanoparticle‐Enhanced Radiation Therapy in In Vitro A549 Lung Carcinoma: Studies in Both Traditional Monolayer and Three Dimensional Cell Culture Models

Purpose:To measure the increase in in vitro radiosensitivity for A549 lung carcinoma cells due to gold nanoparticle (GNP) radiation dose enhancement in both traditional monolayer and three dimensional (3D) cell culture models.Methods:A γH2AX immunofluorescence assay is performed on monolayer A549 cell culture and quantitatively analyzed to measure the increase in double strand breaks (DSBs) resulting from GNP dose enhancement. A clonogenic survival assay (CSA) is then performed on monolayer A549 cell culture to assess true viability after treatment. And lastly, another γH2AX assay is performed on 3D A549 multicellular nodules overlaid on a bed of growth factor reduced matrigel to measure dose response in a model that better recapitulates treatment response to actual tumors in vivo.Results:The first γH2AX assay performed on the monolayer cell culture shows a significant increase in DSBs due to GNP dose enhancement. The maximum average observed increase in normalized fluorescent intensity for monolayer cell culture is 171% for the 6Gy‐treatment groups incubated in 0.556 mg Au/ml solution. The CSA performed on monolayer cell culture also shows considerable GNP dose enhancement. The maximum decrease in the normalized surviving fraction is 12% for the 4Gy‐treatment group incubated in 0.556 mg Au/ml. And lastly, the GNP dose enhancement is confirmed to be mitigated in three dimensional cell culture models as compared to the traditional monolayer model. The maximum average observed dose enhancement for 3D cell culture is 19% for the 6Gy‐treatment groups and incubated in 0.556 mg Au/ml.Conclusion:A marked increase in radiosensitivity is observed for A549 lung carcinoma cells when treated with GNPs plus radiation as opposed to radiation alone. Traditional monolayer cell culture also shows a much more pronounced radiation dose enhancement than 3D cell culture.

Different repair kinetic of DSBs induced by mitomycin C in peripheral lymphocytes of obese and normal weight adolescents

In 2013, 42 million children under the age of 5 years were overweight or obese. In the context of obesity, we recently showed that (1) peripheral lymphocytes of obese children/adolescents had an 8-fold increase in double strand breaks (DSBs), expressed as g-H2AX foci, than normal weight adolescents, and (2) 30% of the damage was retained into chromosome mutations. Thus, we investigated DSBs repair efficiency in a group of obese adolescents assessing the kinetic of H2AX phosphorylation in mitomycin C (MMC)-treated lymphocytes harvested 2 h- or 4 h-post mutagen treatment. According to our previous studies, these harvesting times represent the peak of DSBs induction and the time in which an appreciable DSBs reduction was observed. In addition, we evaluated the expression of the high mobility group box-1 protein (HMGB1), a chromatin remodelling protein involved in DSBs repair and obesity. Compared to normal weight adolescents, obese subjects 1) showed higher levels of g-H2AX foci at either 2 h- (0.239±0.041 vs. 0.473±0.048, P=0.0016) or 4 h- (0.150±0.026 vs. 0.255±0.030, P=0.0198) post mutagen treatment, and 2) have repaired a greater amount of the initial lesions (0.088±0.033 vs. 0.218±0.045, P=0.0408). Concordantly, 1) HMGB1 levels of obese individuals increased and decreased at 2h- or 4 h-post mutagen treatment, respectively, and 2) the opposite occurred for the normal weight adolescents where the protein was down-expressed at 2h and over-expressed at 4h. In conclusion, lymphocytes of obese and normal weight adolescents showed a distinct temporal kinetic of repairing MMC-induced DSBs, together with a different expression of HMGB1. The finding that obesity may modulate the repair of DNA damage induced in lymphocytes by genotoxic agents should be confirmed by further experiments.

Abstract 3064: MicroRNA-21 enhances the effect of ionizing radiation via alteration of the DNA damage response

Abstract Micro-RNA 21 (miR-21) is a well-known onco-miR found to be up-regulated in over 18 major cancer types and is linked to radiation and chemotherapy resistance. It is hypothesized that dysregulation of miR-21 may contribute to treatment resistance through multiple DNA damage response pathways by enhancing DNA repair and increasing DNA damage tolerance. Given its involvement in multiple resistance pathways, miR-21 appears to be an attractive target for overcoming resistance with radiation therapy. In vitro, we demonstrated that knockdown of miR-21 revealed an increase in tumor cell death via clonogenic cell survival curves following exposure to ionizing radiation in multiple cancer cell lines including triple negative breast cancer cells, GBM, and lung cancer. A miR-21 knockout (KO) mouse model was then generated based on deletion of a miR-21 upstream promoter element. To determine if mir-21 KO would lead to radiation sensitivity, miR-21 deficient mice were exposed to 6.5Gy total body irradiation (TBI) and compared to control wild type (WT) mice. miR-21 KO mice died at an average of 12 days after TBI vs. over 100 days survival in wild-type mice. Tissue analysis revealed an increase in double strand breaks measured by elevated γ-H2AX in miR-21 deficient mice. An associated decrease in DNA repair genes such as MSH2 and STAG2 was also seen. Therefore, we hypothesize deletion or silencing of miR-21 results in a radiation sensitive phenotype due to a decreased DNA repair capacity, particularly in the repair of double-strand breaks. In silico analysis predicted miR-21 targets involved in the DNA repair response including rad21 and stag2, which are both members of the cohesin complex. The cohesion complex is a ring-shaped protein complex involved in homologous repair and the DNA damage-response. When exposed to ionizing radiation, rad21 and stag2 expression appears inversely correlated with miR-21 levels in vivo and in vitro. Similarly, in the setting of miR-21 knockdown, rad21 and stag2 expression are increased, suggesting dysregulation in the function of the cohesin complex may contribute to genomic instability and a DNA damage-permissive phenotype. In the current study, we propose a novel mechanism for increasing radiation sensitivity through miRNA-mediated pathways controlling cellular DNA-repair. Future studies investigating miR-21 expression in patients may identify subgroups of patients who are at risk for resistance to standard cytotoxic therapies and may open avenues for targeted therapies involving mir-21 mediated DNA repair pathways. Citation Format: Tu Dan, Ajay Palagani, Tiziana DeAngelis, Sunny Han, Lance Liotta, Richard Pestell, Nicole Simone. MicroRNA-21 enhances the effect of ionizing radiation via alteration of the DNA damage response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3064. doi:10.1158/1538-7445.AM2015-3064

Genistein Protects Hematopoietic Stem Cells against G-CSF–Induced DNA Damage

Granulocyte colony-stimulating factor (G-CSF) has been used to treat neutropenia in various clinical settings. Although clearly beneficial, there are concerns that the chronic use of G-CSF in certain conditions increases the risk of myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML). The most striking example is in severe congenital neutropenia (SCN). Patients with SCN develop MDS/AML at a high rate that is directly correlated to the cumulative lifetime dosage of G-CSF. Myelodysplastic syndrome and AML that arise in these settings are commonly associated with chromosomal deletions. We have demonstrated in this study that chronic G-CSF treatment in mice results in expansion of the hematopoietic stem cell (HSC) population. In addition, primitive hematopoietic progenitors from G-CSF-treated mice show evidence of DNA damage as demonstrated by an increase in double-strand breaks and recurrent chromosomal deletions. Concurrent treatment with genistein, a natural soy isoflavone, limits DNA damage in this population. The protective effect of genistein seems to be related to its preferential inhibition of G-CSF-induced proliferation of HSCs. Importantly, genistein does not impair G-CSF-induced proliferation of committed hematopoietic progenitors, nor diminishes neutrophil production. The protective effect of genistein was accomplished with plasma levels that are attainable through dietary supplementation.

Berberine induces double-strand DNA breaks in Rev3 deficient cells

Berberine is a natural isoquinoline alkaloid, the majority of which is extracted from Huang Lian and other medicinal herbs. Numerous studies have revealed that berberine exhibits anticancer activity, however the mechanisms underlying this effect remain elusive. To examine these mechanisms, we analyzed the effects of berberine on a panel of DNA repair deficient chicken B lymphocyte (DT40) clones. Our results revealed that DT40 cells deficient in Rev3 (Rev3-/-), a translesion DNA synthesis (TLS) gene, were hypersensitive to berberine. Following berberine treatment, cell cycle analysis identified that G2/M arrest was increased in Rev3-/- cells. Furthermore, compared with wild-type cells (WT), berberine also induced a significant increase in double-strand breaks (DSBs) in Rev3-/- cells, as revealed by chromosomal aberration (CA) analysis. These results suggest that berberine is able to induce DNA damage, and that the Rev3 associated DNA repair pathway participates in the processes that aid its repair.

Molecular mechanisms for inhibition of colon cancer cells by combined epigenetic-modulating epigallocatechin gallate and sodium butyrate

Bioactive compounds are considered safe and have been shown to alter genetic and epigenetic profiles of tumor cells. However, many of these changes have been reported at molecular concentrations higher than physiologically achievable levels. We investigated the role of the combinatorial effects of epigallocatechin gallate (EGCG), a predominant polyphenol in green tea, and sodium butyrate (NaB), a dietary microbial fermentation product of fiber, in the regulation of survivin, which is an overexpressed anti-apoptotic protein in colon cancer cells. For the first time, our study showed that the combination treatment induced apoptosis and cell cycle arrest in RKO, HCT-116 and HT-29 colorectal cancer cells. This was found to be regulated by the decrease in HDAC1, DNMT1, survivin and HDAC activity in all three cell lines. A G2/M arrest was observed for RKO and HCT-116 cells, and G1 arrest for HT-29 colorectal cancer cells for combinatorial treatment. Further experimentation of the molecular mechanisms in RKO colorectal cancer (CRC) cells revealed a p53-dependent induction of p21 and an increase in nuclear factor kappa B (NF-κB)-p65. An increase in double strand breaks as determined by gamma-H2A histone family member X (γ-H2AX) protein levels and induction of histone H3 hyperacetylation was also observed with the combination treatment. Further, we observed a decrease in global CpG methylation. Taken together, these findings suggest that at low and physiologically achievable concentrations, combinatorial EGCG and NaB are effective in promoting apoptosis, inducing cell cycle arrest and DNA-damage in CRC cells.

ERG induces a mesenchymal-like state associated with chemoresistance in leukemia cells

Overexpression of the oncogene ERG (ETS-related gene) is an adverse prognostic factor in acute myeloid and T-cell lymphoblastic leukemia (AML and T-ALL). We hypothesize that ERG overexpression is associated with primary drug resistance thereby influencing the outcome in leukemia. We previously reported a cell-line based model of ERG overexpression which induced a potentially chemo-resistant spindle shape cell type. Herein, we report a specific transcriptional gene signature for the observed spindle shaped morphology. Genes significantly over-expressed after ERG induction strongly resembled adhesive mesenchymal-like genes that included integrins (ITGA10, ITGB5, ITGB3, ITGA2B), CD44, and CD24. Interestingly, the mesenchymal-like signature was accompanied by the repression of DNA chromatin remodeling and DNA repair genes, such as CHEK1, EZH2, SUZ12, and DNMT3a. The ERG-induced mesenchymal-like signature positively correlated with TMPRSS2-ERG prostate tissues and invasive breast cancer mRNA expression datasets reflecting a general ERG-driven pattern of malignancy. Furthermore, inhibitors modulating ERG druggable pathways WNT, PKC, and AKT, and chemotherapeutic agent cytarabine revealed ERG-induced drug resistance. In particular, PKC412 treatment enhanced proliferative rates and promoted spindle shape formation in ERG-induced cells. Nilotinib and dasatinib were effective at abolishing ERG-induced cells. Moreover, ERG overexpression also led to an increase in double strand breaks. This report provides mechanistic clues into ERG-driven drug resistance in the poor prognostic group of high ERG expressers, provides insight to improved drug targeted therapies, and provides novel markers for a mesenchymal-like state in acute leukemia.

Combination therapies in Myeloproliferative Neoplasms: why do we need them and how to identify potential winners?

The myeloproliferative neoplasms (MPN) are clonal myeloid disorders characterized by proliferation of mature myeloid cells, such that in polycythaemia vera (PV), the red cell proliferation dominates, platelets in essential thrombocythaemia (ET) and in myelofibrosis (MF), there may be cytopenia or proliferation, but the characteristic feature is the strikingly abnormal bone marrow stroma. These entities have a tendency to show phenotypic mimicry and may transform from one to another, for example, 20–30% of patients with PV are likely to develop MF. The significant event in this field was the recognition that Janus Kinase-2 (JAK2) activation was highly prevalent, followed by the description of the JAK2V617F mutation in 2005 (vide infra), which stimulated renewed interest in disease biology. Janus Kinase-2-targeted therapies have led to marked improvements for patients with this condition. However, it is obvious that the pathogenesis of these complex disorders reaches beyond this mutation; only 50–60% of patients with ET, for example, have the JAK2 mutation and several additional mutations have been described, which are of relevance in both the pathogenesis and clinical phenotype of these conditions.

Abstract LB-393: Genotoxicity of arsenic in primary human bronchial cells

Abstract Arsenic originates from both geochemical and numerous anthropogenic activities including mining, combustion of fossil fuels, wood preservation, agriculture and metallurgy. Exposure of the general public to significant levels of arsenic is widespread. Arsenic is a well-documented human carcinogen. Long-term exposure to low levels of arsenic in drinking water have been linked to bladder, lung, kidney, liver, prostate, and skin cancer. Among them, lung cancer is of great public concern. However, little is known about how arsenic causes lung cancer. The purpose of this study was to determine the cytotoxicity and genotoxicity in human primary bronchial fibroblasts cells (NHBF). Our data show that arsenic induces a concentration-dependent increase in cell death after acute (24 h) or chronic (120 h) exposure. 0.1, 1 and 10 uM arsenic for 24 h induced 85, 64 and 35 percent relative survival, respectively, and induced 73, 57 and 11 percent of relative survival, respectively after 120 h treatment. 24 h exposure of 5 and 10 uM arsenic induced 15 and 32 chromosome damage compared to the control. Comet assay was used to measure the formation of DNA double strand breaks. We found that 120 h exposure induced a concentration-dependent increase in double strand breaks. Future study will focus on arsenic cytotoxicity and genotoxicity in primary bronchial epithelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-393. doi:1538-7445.AM2012-LB-393

B-Myb is Critical for Proper DNA Duplication During an Unperturbed S Phase in Mouse Embryonic Stem Cells

A common feature of early embryo cells from the inner cell mass (ICM) and of ESCs is an absolute dependence on an atypical cell cycle in which the G1 phase is shortened to preserve their self-renewing and pluripotent nature. The transcription factor B-Myb has been attributed a role in proliferation, in particular during the G2/M phases of the cell cycle. Intriguingly, B-Myb levels in ICM/ESCs are greater than 100 times compared with those in normal proliferating cells, suggesting a particularly important function for this transcription factor in pluripotent stem cells. B-Myb is essential for embryo development beyond the preimplantation stage, but its role in ICM/ESCs remains unclear. Using a combination of mouse genetics, single DNA fiber analyses and high-resolution three-dimensional (3D) imaging, we demonstrate that B-Myb has no influence on the expression of pluripotency factors, but instead B-Myb ablation leads to stalling of replication forks and superactivation of replication factories that result in disorganization of the replication program and an increase in double-strand breaks. These effects are partly due to aberrant transcriptional regulation of cell cycle proliferation factors, namely c-Myc and FoxM1, which dictate normal S phase progression. We conclude that B-Myb acts crucially during the S phase in ESCs by facilitating proper progression of replication, thereby protecting the cells from genomic damage. Our findings have particular relevance in the light of the potential therapeutic application of ESCs and the need to maintain their genomic integrity.

Repair system for noncanonical purines in Escherichia coli.

Exposure of Escherichia coli strains deficient in molybdopterin biosynthesis (moa) to the purine base N-6-hydroxylaminopurine (HAP) is mutagenic and toxic. We show that moa mutants exposed to HAP also exhibit elevated mutagenesis, a hyperrecombination phenotype, and increased SOS induction. The E. coli rdgB gene encodes a protein homologous to a deoxyribonucleotide triphosphate pyrophosphatase from Methanococcus jannaschii that shows a preference for purine base analogs. moa rdgB mutants are extremely sensitive to killing by HAP and exhibit increased mutagenesis, recombination, and SOS induction upon HAP exposure. Disruption of the endonuclease V gene, nfi, rescues the HAP sensitivity displayed by moa and moa rdgB mutants and reduces the level of recombination and SOS induction, but it increases the level of mutagenesis. Our results suggest that endonuclease V incision of DNA containing HAP leads to increased recombination and SOS induction and even cell death. Double-strand break repair mutants display an increase in HAP sensitivity, which can be reversed by an nfi mutation. This suggests that cell killing may result from an increase in double-strand breaks generated when replication forks encounter endonuclease V-nicked DNA. We propose a pathway for the removal of HAP from purine pools, from deoxynucleotide triphosphate pools, and from DNA, and we suggest a general model for excluding purine base analogs from DNA. The system for HAP removal consists of a molybdoenzyme, thought to detoxify HAP, a deoxyribonucleotide triphosphate pyrophosphatase that removes noncanonical deoxyribonucleotide triphosphates from replication precursor pools, and an endonuclease that initiates the removal of HAP from DNA.

Mechanisms of radiosensitization in iododeoxyuridine-substituted cells.

The radiosensitization caused by iododeoxyuridine (IdU)-substitution of thymidine in V79-171 cells is decreased by the presence of acetone during irradiation. Acetone, at 1 mol dm-3, removes almost all the increase in double strand breaks (dsbs) caused by IdU substitution, but removes only about two-thirds of the enhancement in killing. Similar observations were made with BrdU-substituted cells. The decrease in cell radiosensitization coincides with the removal of the additional dsbs. The protection afforded by acetone is assumed to be due to its scavenging of hydrated electrons, thought to be the active species causing enhanced DNA damage in the presence of halogenated pyrimidines. The residual component of IdU radiosensitization, which could not be removed by treatment with acetone, is manifest largely as a shoulder effect (Dq) and may be due to either a subset of non-scavengable, lethal dsbs and/or the influence of IdU on the fixation of potentially lethal damage. This study further demonstrates that halogenated pyrimidine-mediated radiosensitization consists of at least distinct components each associated with a different phenomenon.